CN109239113B - The transmission electron microscope sample preparation method of the Armco magnetic iron protein nano particle of bio-mimetic syntheses - Google Patents
The transmission electron microscope sample preparation method of the Armco magnetic iron protein nano particle of bio-mimetic syntheses Download PDFInfo
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- CN109239113B CN109239113B CN201811156860.9A CN201811156860A CN109239113B CN 109239113 B CN109239113 B CN 109239113B CN 201811156860 A CN201811156860 A CN 201811156860A CN 109239113 B CN109239113 B CN 109239113B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
- G01N23/2202—Preparing specimens therefor
Abstract
The invention discloses a kind of transmission electron microscope sample preparation methods of the Armco magnetic iron protein nano particle of bio-mimetic syntheses comprising: the buffer of the Armco magnetic iron protein nano particle of bio-mimetic syntheses is mixed with deionized water, and is diluted to suitable concentration；To the mixed liquor cryostat ultrasonic vibration, well-mixed sample suspension is obtained；Electrostatic precipitation processes are carried out to ultrathin carbon films support grid；Wellability processing is carried out to the ultrathin carbon films support grid again；Treated that ultrathin carbon films support grid is placed on clean filter paper by described, to sample suspension described in its surface titration；The ultrathin carbon films support grid for carrying the sample suspension is dry；It is taken out after the sample is completely dried, as Armco magnetic iron protein nano particle transmission electron microscope sample.The present invention is able to solve transmission electron microscope sample preparation difficulty, inefficiency and the sordid problem of ultrathin carbon films support grid back end of Armco magnetic iron protein nano particle, and can present well to the intrinsic monodispersity state of sample.
Description
Technical field
The present invention relates to a kind of transmission electron microscope sample preparation methods of the Armco magnetic iron protein nano particle of bio-mimetic syntheses, belong to
The transmission electron microscope sample preparation field of ferritin nano particle.
Background technique
The Armco magnetic iron protein nano particle of bio-mimetic syntheses is the biomineralization albumen with gene engineering expression --- recombined human H
Subunit ferritin is as mineralising template, and according to the biomineralization principle of ferritin in human body, one kind of bio-mimetic syntheses has typical case
The magnetic recombinant human iron albumen of core-shell type structure includes a nanometer monocrystalline magnetic iron ore (Fe in each ferritin shell3O4)
Particle.The Armco magnetic iron protein nano particle of bio-mimetic syntheses usually has uniform monodispersity, shape, size tunable, weak magnetic mutual
The characteristics such as effect, and the activity of the peroxidase of magnetic ferritin is increased as partial size increases, and can be used for tumor tissues
Immunohistochemical staining and rapid pathological diagnosis.People is similar to since the magnetic recombinant human iron albumen of this bio-mimetic syntheses has
Ferritin shell in body can penetrate a variety of barriers in organism, be enriched in tumor lesion area, and iron core is magnetic strong Asia
Ferromagnetic mineral has had both biocompatibility and the good feature of monodispersity, thus in materialogy, biomedicine and rock magnetism
Field has important application value.
The Armco magnetic iron protein nano grain diameter of bio-mimetic syntheses is all minimum, about in 2~8nm, needs after the completion of preparation pair
Its monodispersity, shape, size, ingredient and crystal structure carry out comprehensive characterization and analysis, could go deep into this way
Recognize the structure and performance of the Armco magnetic iron protein nano particle of bio-mimetic syntheses, effective application for its next step in each field is carried out
Place mat also provides new method and technical foundation for the optimization of further performance and the improvement of synthetic parameters.Since its size is several
A nanometer of scope, characterization and the analysis that micro-scale is carried out to it are then relatively difficult.Currently, transmission electron microscope
(Transmission Electron Microscopy, abbreviation TEM) has excellent spatial resolution (spherical aberration correction transmission electricity
The point resolution of mirror is up to 0.05nm), it can pattern to Armco magnetic iron protein nano particle, size distribution, ingredient and high-resolution
Structure is analyzed and characterized.Transmission electron microscope is using electron beam as light source, e-beam projection that is aggregated and accelerating to thin sample
On, high energy electron and sample interact, and generate various physical signals, and wherein incident electron is taken through the part electronics of sample
The band extrinsic information of sample, due to the thickness of each microcell of sample, atomic number, crystal structure and position to etc. differences, then
Transmitted electron angle of scattering across sample is different, to be formed, light and shade is different, reflects the image of sample message.
Carry out Armco magnetic iron protein nano particle transmission electron microscope analysis characterization, to obtain it is good, reflection sample it is intrinsic
The images of transmissive electron microscope of information, preparing for sample are most important.But the preparation process of transmission electron microscope sample is complex, there is
Many uncertainties, such as thickness of sample, sample distribution quantity, impurity pollution, the clean level etc. of support grid back end.Especially
Only in the Armco magnetic iron protein nano particle of several nanometers of bio-mimetic syntheses, the preparation of transmission electron microscope sample requires more partial size
It is high.
Conventional transmission electron microscope sample preparation method is directly to drip the aqueous solution containing Armco magnetic iron protein nano particle
In ultrathin carbon films support grid, transmission electron microscope observing is carried out after to be dried.The transmission electron microscope sample poor effect of this method preparation, tool
1. body is showed themselves in that since ultrathin carbon films support grid is to cover the magnificent film in one layer of side on metal grill, then spray in the magnificent film upper surface in side
It plates one layer of carbon film and is formed, its surface is easy to that electrostatic adsorption occurs in air, to be formed in ultrathin carbon films micro-
Grain pollution so that back end is not clean, and then influences the transmission electron microscope observing effect of Armco magnetic iron protein nano particle；2. due to ultra-thin
For carbon film in hydrophobic, wellability is poor, if copper mesh is completely covered in the sample solution of titration, due to Armco magnetic iron protein nano granular mass
It is small, it is suspended on drop, finally as aqueous solution leaves ultrathin carbon films or only a small amount of residual, causes transmission electron microscope observing
When to find sample difficult, be in most cases that can not find sample；If only dripping a small amount of sample drop no more than ultra-thin carbon
When the diameter of film copper mesh, then because the hydrophobicity of ultrathin carbon films makes sample solution remain a droplet-like, and then the table of drop
Face tension causes Armco magnetic iron protein nano particle agglomeration, will be unable to carry out its form, the transmission of crystallite dimension and full resolution pricture
Electronic Speculum characterization, and the quality of its intrinsic dispersibility also suffers from influence；3. titrating good sample usually in air or anaerobism
It is spontaneously dried in case, needs etc. several hours, it is extremely low to prepare sample efficiencies, and the too long sample of drying process can gather
Collection, can not reflect the quality of intrinsic monodispersity.
Therefore, using conventional sample preparation methods, can Armco magnetic iron protein nano particle to bio-mimetic syntheses monodisperse
Property, uniform particle diameter degree, particle size and full resolution pricture etc. characterization bring very big problem, and then influence its structure with
The performance optimization of the characterization and next step of performance and synthetic technology are improved, and the magnetic ferritin of bio-mimetic syntheses will eventually be hindered
The extensive use of nano particle.
Summary of the invention
In view of this, the present invention is intended to provide a kind of transmission electron microscope sample of the Armco magnetic iron protein nano particle of bio-mimetic syntheses
Preparation method, the transmission electron microscope sample that this method can solve Armco magnetic iron protein nano particle prepare difficult, inefficiency and surpass
The thin sordid problem of carbon film support grid back end, and the quality of the intrinsic monodispersity of sample can be presented well.
The present invention provides a kind of transmission electron microscope sample preparation method of the Armco magnetic iron protein nano particle of bio-mimetic syntheses,
Include:
S1: the buffer of the Armco magnetic iron protein nano particle of bio-mimetic syntheses is mixed with deionized water, and is diluted to properly
Concentration；
S2: to the mixed liquor cryostat ultrasonic vibration, well-mixed sample suspension is obtained；
S3: electrostatic precipitation processes are carried out to ultrathin carbon films support grid；
S4: wellability processing is carried out to the ultrathin carbon films support grid again；
S5: treated that ultrathin carbon films support grid is placed on clean filter paper by described, outstanding to sample described in its surface titration
Supernatant liquid；
S6: the ultrathin carbon films support grid for carrying the sample suspension is dry；
S7: taking out after the sample is completely dried, as Armco magnetic iron protein nano particle transmission electron microscope sample.
The preparation method according to the present invention, using the deionized water by the Armco magnetic iron protein nano of bio-mimetic syntheses in S1
The buffer of particle is diluted to 0.1-0.2mg/ml.It BCA kit can be used to carry out concentration mensuration to magnetic ferritin, walk in detail
Rapid visible kit explanation.
Further, the purity of the deionized water is that resistivity reaches 18.2M Ω cm, to prevent the cation in water right
The Armco magnetic iron protein nano particulate samples of bio-mimetic syntheses pollute.
The preparation method according to the present invention carries out cryostat to the mixed liquor in centrifuge tube using ultrasonoscope in S2
Ultrasonic vibration is cooled down when the cryostat ultrasound, ultrasonic time 5-8min, ultrasonic power 80W using ice cube.
The preparation method according to the present invention, ultrathin carbon films support grid described in S3 are nickel screen ultrathin carbon films, the ultra-thin carbon of molybdenum net
Film or tungsten net ultrathin carbon films, the preferred nickel screen ultrathin carbon films of the present invention.
The preparation method according to the present invention, in S3 using ion blower Xelminator to the ultrathin carbon films support grid into
Row goes electrostatic precipitation processes, to remove the pollution of Electrostatic Absorption in support grid, is generated by the ion blower Xelminator big
Amount has the Tiny pore of positive and negative charge, blows to ultrathin carbon films support grid surface, neutralizes the charge of institute's band in support grid.
Further, the ultrathin carbon films support grid is placed in glass culture dish, apart from ion blower Xelminator 20~
40cm, Boiler pressure control 0.55m3/ min, neutralization time are 5~20s, then overturn ultrathin carbon films support grid another side court with tweezers
On, carry out identical operation.
The ultrathin carbon films support grid is carried out wellability processing in S4 by the preparation method according to the present invention, can be very
Good adheres to the sample solution, is reacted using active O free radical with the ultrathin carbon films support grid surface active, forms infiltration
The good ultrathin carbon films support grid of property.
Further, wellability processing is carried out to the ultrathin carbon films support grid using UV ozone machine, wellability processing
The producing method of active O free radical is the ultraviolet light and O of shorter wavelength2Effect generate ozone molecule, ozone again with longer wavelength
Action of ultraviolet light decompose generate activity O free radical.
Alternatively, further, carrying out wellability processing, institute to the ultrathin carbon films support grid using plasma rf power supply
State Ar, O that plasma rf power supply will be passed through2Mixed gas ionizes, later O2The electronic action formed with ionization forms activity
O free radical.
The preparation method according to the present invention sucks the sample suspension titration described ultra-thin using pipettor in S5
The surface of carbon film, pipettor suck suspension at least 10 μ l, and when titration makes sample suspension that entire ultrathin carbon films be completely covered to carry
Net, so that the sample suspension tiles naturally, it is ensured that the intrinsic monodispersity state of sample, i.e., single magnetic ferritin are received
Boundary does not contact between rice grain, does not reunite.
The ultrathin carbon films support grid for carrying the sample suspension is put into vacuum in S6 by the preparation method according to the present invention
It is dry in drying box, comprising: the ultrathin carbon films support grid for carrying the sample suspension is moved into glass culture dish, is closed the lid
Then son is put into togerther in vacuum oven dry together with culture dish.
Further, vacuum degree < 10 in the vacuum oven-3Pa, heating temperature≤60 DEG C, preferably 60 DEG C are done
Dry 30~40min of time.
Compared with the prior art, preparation method of the present invention has the advantage that
1) preparation method of the present invention can be removed in support grid by carrying out electrostatic precipitation processes to ultrathin carbon films support grid
The pollutant of Electrostatic Absorption purifies support grid back end, improves the transmission electron microscope observing effect of Armco magnetic iron protein nano particle, favorably
In the microscopic morphology of sample and the characterization of high resolution structure, and then improve transmission electron microscope conventional efficient.
2) preparation method according to the present invention carries out wellability processing to ultrathin carbon films support grid, so that the sample of titration
Solution covers support grid naturally, avoids Armco magnetic iron protein nano particle because quality is small as suspension is detached from asking for ultrathin carbon films
Topic, Armco magnetic iron protein nano particle agglomeration caused by also eliminating because of Surface Tension of Liquid Drops are conducive to intrinsic point that keeps sample
Dissipate property, and then be conducive to the form of Armco magnetic iron protein nano particle, crystallite dimension and full resolution pricture and intrinsic dispersibility it is saturating
Radio mirror characterization.
3) preparation method of the present invention by sample carry out vacuum and heating drying, improve sample drying efficiency,
Conducive to the intrinsic monodispersity state for keeping sample.
4) transmission electron microscope sample of the bio-mimetic syntheses Armco magnetic iron protein nano particle of the method preparation according to the present invention
Success rate is high, and preparation efficiency is high, the transmission electron microscope sample preparation of the Armco magnetic iron protein nano particle suitable for different nanoscales.
Detailed description of the invention
Fig. 1 is the transmission electron microscope sample preparation flow figure of the Armco magnetic iron protein nano particle of bio-mimetic syntheses of the present invention.
Fig. 2 is the Armco magnetic iron protein nano particle (P-MHFn- of the bio-mimetic syntheses prepared according to the method for the invention
5000) transmission electron microscope photo.
Fig. 3 is the High-Resolution Map of the Armco magnetic iron protein nano particle of the bio-mimetic syntheses prepared according to the method for the invention
Picture.
Fig. 4 is that the monocrystalline electronics of the Armco magnetic iron protein nano particle of the bio-mimetic syntheses prepared according to the method for the invention spreads out
Penetrate map.
Fig. 5 is the composition information of the Armco magnetic iron protein nano particle of the bio-mimetic syntheses prepared according to the method for the invention.
Fig. 6 is the Armco magnetic iron protein nano particle (E-MHFn-800) of the bio-mimetic syntheses prepared according to the method for the invention
Transmission electron microscope photo.
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
Unless otherwise specified, various raw material, reagent, the instrument and equipment etc. used in the present invention, can pass through
Market is commercially available or can be prepared by existing method.
A kind of method of the transmission electron microscope sample of the Armco magnetic iron protein nano particle of the present invention for preparing bio-mimetic syntheses has
Body the following steps are included:
S1: the buffer of the Armco magnetic iron protein nano particle of bio-mimetic syntheses is mixed with deionized water, and is diluted to properly
Concentration；
Magnetic liquor ferri albuminati centrifugation 10min, the revolving speed 12000r/min for taking suitable biomimetic mineralization synthesis, remove outstanding
Floating impurity in the solution.Concentration mensuration, the visible kit explanation of detailed step are carried out to magnetic ferritin with BCA kit.
The Armco magnetic iron protein nano particle buffer of known concentration is put into centrifuge tube again, adds going for corresponding amount according to a certain percentage
Ionized water, is diluted to the visible pico- yellowing of solution naked eyes, and the solution concentration is 0.1-0.2mg/ml.
Further, the purity for the deionized water of diluted magnetic ferritin nano particle buffer reaches for resistivity
18.2M Ω cm, to prevent the cation in water from polluting to the Armco magnetic iron protein nano particulate samples of bio-mimetic syntheses.
S2: to the mixed liquor cryostat ultrasonic vibration, well-mixed sample suspension is obtained；
Specifically, it is cooled down when cryostat ultrasonic vibration using ice cube, prevents sample in ultrasonic procedure ultrasonic by thermal oxide
Time is 5~8min, ultrasonic power 80W.
S3: electrostatic precipitation processes are carried out to ultrathin carbon films support grid；
Specifically, using ion blower Xelminator, the Tiny pore for largely having positive and negative charge is generated, ultra-thin carbon is blowed to
Film support grid surface neutralizes the charge of institute's band in support grid.
Further, ultrathin carbon films support grid is placed in glass culture dish, is placed on apart from ion blower Xelminator 20
The position of~40cm, Boiler pressure control 0.55m3/ min, neutralization time are 5~20s, then overturn ultrathin carbon films support grid with tweezers
It is another face-up, carry out the above identical operation.When support grid surface is negatively charged, it absorbs the positive charge in air-flow, works as load
When net surface is positively charged, it absorbs the negative electrical charge in air-flow, is neutralized the electrostatic on surface, subsidiary blowing function, thus
Achieve the purpose that eliminating electrostatic blows fume off.
In the present embodiment, ultrathin carbon films support grid is placed in glass culture dish, and ultrathin carbon films support grid and ion is set
The suitable distance of blower Xelminator and the air quantity of ion blower, it can be ensured that effectively eliminate the micro- of Electrostatic Absorption in support grid
While grain pollutant, support grid is not blown away by band electric souffle.
In this step, since the Armco magnetic iron protein nano grain diameter of bio-mimetic syntheses is usually 2-8nm, it is carried out
The thickness of ultrathin carbon films support grid answers < 7nm when prepared by transmission electron microscope sample, is received with preventing ultrathin carbon films back end from influencing magnetic ferritin
The micro-imaging of rice grain sample.
Further, the ultrathin carbon films support grid can be nickel screen ultrathin carbon films, be also possible to molybdenum net ultrathin carbon films, tungsten net
Ultrathin carbon films.The Mohs' hardness of nickel screen, molybdenum net and tungsten net is respectively 4,5.5,7.5, higher than copper mesh (Mohs' hardness 2.5~3),
Therefore relative to copper mesh ultrathin carbon films, using nickel screen, molybdenum net and tungsten net ultrathin carbon films support grid when it is less likely to occur to deform, keep away
The transmission electron microscope observing that deformation causes ultrathin carbon films to crimp and influences 2-8nm sample is exempted from.The ultra-thin carbon of the preferred nickel screen of the present invention
Film.
S4: wellability processing is carried out to the ultrathin carbon films support grid again；
Specifically, the ultraviolet light for the use of UV ozone cleaning machine generation wavelength being 185nm and 254nm, the purple of shorter wavelength
Outer light and O2Effect generates ozone molecule, and ozone decomposes with the action of ultraviolet light of longer wavelength generate activity O free radical again, then with
Ultrathin carbon films support grid surface active reacts to form the good support grid of wellability.
Alternatively, support grid is put into plasma cleaning instrument, it is passed through Ar, O2Mixed gas, Ar and O2Ratio is 75%:
25%, mixed gas is ionized by radio-frequency power supply, then O2The electronic action formed with ionization forms active O free radical, then
It reacts to form the good support grid of wellability with ultrathin carbon films support grid surface active.
S5: treated that ultrathin carbon films support grid is placed on clean filter paper by described, outstanding to sample described in its surface titration
Supernatant liquid；
Specifically, the 10 μ l of suspension of pipettor sucking, 2~3 drop of drop, when titration, ensure that sample solution is completely covered entirely
Ultrathin carbon films support grid, so that suspension tiles naturally, the dispersed state for keeping sample intrinsic.
S6: the ultrathin carbon films support grid for carrying the sample suspension is dry；
Specifically, first the support grid for carrying sample is moved into glass culture dish, is closed the lid, then together with culture dish
It is put into drying in vacuum oven, so that it is guaranteed that support grid is not siphoned away by vacuum.Vacuum degree < 10 in vacuum oven-3Pa, heating
Temperature≤60 DEG C, optimized temperature are 60 DEG C, drying time 30min.
S7: taking out after the sample is completely dried, as Armco magnetic iron protein nano particle transmission electron microscope sample.
It should be noted that the transmission electron microscope sample prepared should be put into the form that sample is carried out in transmission electron microscope as early as possible,
Crystallite dimension, ingredient, full resolution pricture and intrinsic dispersibility characterization.Sample should be put into vacuum oven when saving, with
Exempt to be contaminated.
Embodiment 1
The recombined human H subunit ferritin mineralising of Pichia anomala expression is synthesized into 5000 resulting magnetism of iron atom of iron
Ferritin nano particle (P-MHFn-5000) buffer carries out determination of protein concentration with BCA kit, takes a certain amount of be put into
In 1.5ml centrifuge tube, it is proportionally added into the deionized water that resistivity reaches 18.2M Ω cm, being diluted to concentration is 0.1mg/
The mixed liquor of ml, the visible pico- yellowing of solution of naked eyes；Using ultrasonoscope to the mixed liquor cryostat ultrasonic vibration in centrifuge tube,
Ice cube is added in ultrasonic wave to be cooled down, ultrasonic time 10min obtains well-mixed sample suspension；Choosing diameter is
3mm, thickness < 7nm nickel screen ultrathin carbon films support grid；Nickel screen ultrathin carbon films support grid is gone using ion blower Xelminator
Electrostatic precipitation processes, ultrathin carbon films support grid are placed in glass culture dish, apart from ion blower Xelminator 30cm, Boiler pressure control
For 0.55m3/min, time 10s is neutralized, it is then another up with tweezers overturning ultrathin carbon films support grid, it carries out above identical
Operation；Ultrathin carbon films support grid is put into UV ozone cleaning machine again, work is generated by ultraviolet light and the O2 effect of different wave length
Property O free radical, and then it is reacted with ultrathin carbon films support grid surface active, forms the good ultrathin carbon films support grid of wellability；It will go quiet
The clean ultrathin carbon films support grid that electric precipitation processing and wellability are handled well is placed on clean filter paper, sucks 10 μ l with pipettor
Sample suspension toward support grid surface titration, 2~3 drop of drop, when titration, ensures that sample solution is completely covered entire ultrathin carbon films and carries
Net, so that suspension tiles naturally, the monodispersity state for keeping sample intrinsic；The support grid for carrying sample is finally moved into glass
In culture dish, it then is put into togerther drying in vacuum oven together with culture dish, so that it is guaranteed that support grid is not siphoned away by vacuum, vacuum
Vacuum degree < 10-3Pa in drying box, heating temperature are 60 DEG C, drying time 30min；It is taken out after sample is completely dried,
Armco magnetic iron protein nano particle transmission electron microscope sample as observation.
Armco magnetic iron protein nano particle transmission electron microscope sample is put into the shape that P-MHFn-5000 sample is carried out in transmission electron microscope
State, crystallite dimension, ingredient, full resolution pricture and intrinsic monodispersity characterization.
Fig. 2 is the transmission electron microscope bright field image photo of the Armco magnetic iron protein nano particle (P-MHFn-5000) of bio-mimetic syntheses, right
Figure is it is found that the intrinsic dispersibility of the Armco magnetic iron protein nano particle is fine, and shape is uniform spherical shape, and average grain diameter is about 4.5
±1.2nm。
Fig. 3 is the full resolution pricture of single Armco magnetic iron protein nano particle, shows clearly lattice dot matrix, it is seen that it is tied
Brilliant degree is very good.
Fig. 4 is the monocrystalline electronic diffraction map of the Armco magnetic iron protein nano particle, can reflect the crystal of ferritin kernel
Structural information, determining its crystal structure such as icon is magnetic iron ore (Fe3O4).
Fig. 5 is the fingerprints of the Armco magnetic iron protein nano particle, main component Fe, O.
Embodiment 2
By the recombined human H subunit ferritin mineralising synthesis obtained using prokaryotic expression system, (synthetic reaction pH is controlled
8.5, temperature is 75 DEG C) into resulting Armco magnetic iron protein nano particle (E-MHFn-800) buffer of 800 iron atoms of iron, it adopts
Determination of protein concentration is carried out with BCA kit, is diluted to M-HFn with the deionized water of resistivity up to 18.2M Ω cm
0.1mg/mL sample to be tested；Using ultrasonoscope to the mixed liquor cryostat ultrasonic vibration in centrifuge tube, ice is added in ultrasonic wave
Block is cooled down, and ultrasonic time 10min obtains well-mixed sample suspension；Selection diameter is 3mm, thickness < 7nm nickel
Net ultrathin carbon films support grid；Electrostatic precipitation processes are carried out to nickel screen ultrathin carbon films support grid using ion blower Xelminator, are surpassed
Thin carbon film support grid is placed in glass culture dish, apart from ion blower Xelminator 30cm, Boiler pressure control 0.55m3/min, in
It is then another up with tweezers overturning ultrathin carbon films support grid with time 10s, carry out the above identical operation；Support grid is put again
Enter in plasma cleaning instrument, be passed through 75%Ar gas and the mixed gas of 25%O2, is ionized mixed gas by radio-frequency power supply
Activity O free radical is formed, then reacts to form the good support grid of wellability with ultrathin carbon films support grid surface active；It will go at electrostatic precipitation
The clean ultrathin carbon films support grid that reason and wellability have been handled is placed on clean filter paper, and the sample for sucking 10 μ l with pipettor is outstanding
Supernatant liquid is toward support grid surface titration, 2~3 drop of drop, and when titration ensures that entire ultrathin carbon films support grid is completely covered in sample solution, so that outstanding
Supernatant liquid tiles naturally, the monodispersity state for keeping sample intrinsic；Finally the support grid for carrying sample is moved into glass culture dish,
Then it is put into togerther drying in vacuum oven together with culture dish, so that it is guaranteed that support grid is not siphoned away by vacuum, in vacuum oven
Vacuum degree < 10-3Pa, heating temperature be 60 DEG C, drying time 30min；It is taken out after sample is completely dried, becomes observation
Armco magnetic iron protein nano particle transmission electron microscope sample.
Armco magnetic iron protein nano particle transmission electron microscope sample is put into and carries out the aobvious of E-MHFn-800 sample in transmission electron microscope
Microanalysis characterization.
Fig. 6 is the transmission electron microscope bright field image photo of the Armco magnetic iron protein nano particle (E-MHFn-800) of bio-mimetic syntheses, right
Figure it is found that the Armco magnetic iron protein nano particle be rendered as it is intrinsic dispersibility very well, do not assemble mutually between particle, shape is equal
One spherical shape, average grain diameter is in 1.4 ± 0.5nm.
Although preferred embodiments of the present invention have been described, it is created once a person skilled in the art knows basic
Property concept, then additional changes and modifications may be made to these embodiments.So it includes excellent that the following claims are intended to be interpreted as
It selects embodiment and falls into all change and modification of the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (12)
1. a kind of transmission electron microscope sample preparation method of the Armco magnetic iron protein nano particle of bio-mimetic syntheses comprising:
S1: the buffer of the Armco magnetic iron protein nano particle of bio-mimetic syntheses is mixed with deionized water, and is diluted to suitable dense
Degree；
S2: to the mixed liquor cryostat ultrasonic vibration, well-mixed sample suspension is obtained；
S3: electrostatic precipitation processes are carried out to ultrathin carbon films support grid；
S4: wellability processing is carried out to the ultrathin carbon films support grid again；
S5: treated that ultrathin carbon films support grid is placed on clean filter paper by described, to sample suspension described in its surface titration；
S6: the ultrathin carbon films support grid for carrying the sample suspension is dry；
S7: taking out after the sample is completely dried, as Armco magnetic iron protein nano particle transmission electron microscope sample.
2. preparation method as described in claim 1, which is characterized in that use the deionized water by the magnetic of bio-mimetic syntheses in S1
The buffer of property ferritin nano particle is diluted to 0.1-0.2mg/ml.
3. preparation method as claimed in claim 2, which is characterized in that the purity of the deionized water is that resistivity reaches 18.2M
Ω·cm。
4. preparation method as described in claim 1, which is characterized in that using ultrasonoscope to described mixed in centrifuge tube in S2
It closes liquid and carries out cryostat ultrasonic vibration, cooled down using ice cube when the cryostat ultrasound, ultrasonic time is 5~8min, ultrasonic function
Rate is 80W.
5. preparation method as described in claim 1, which is characterized in that ultrathin carbon films support grid described in S3 are the ultra-thin carbon of nickel screen
Film, molybdenum net ultrathin carbon films or tungsten net ultrathin carbon films.
6. preparation method as claimed in claim 5, which is characterized in that the ultrathin carbon films support grid is nickel screen ultrathin carbon films.
7. preparation method as described in claim 1, which is characterized in that using ion blower Xelminator to described super in S3
Thin carbon film support grid carries out electrostatic precipitation processes.
8. the preparation method as described in any claim in claim 1-7, which is characterized in that the ultrathin carbon films support grid is set
In in glass culture dish, apart from ion blower 20~40cm of Xelminator, Boiler pressure control 0.55m3/ min, neutralize the time be
5~20s, it is then another up with tweezers overturning ultrathin carbon films support grid, carry out identical operation.
9. preparation method as described in claim 1, which is characterized in that use UV ozone cleaning machine or plasma in S4
Radio-frequency power supply carries out wellability processing to the ultrathin carbon films support grid, utilizes active O free radical and the ultrathin carbon films support grid table
Face priming reaction forms the good ultrathin carbon films support grid of wellability.
10. preparation method as described in claim 1, which is characterized in that the ultra-thin carbon of the sample suspension will be carried in S6
Film support grid is put into vacuum oven dry, comprising: the ultrathin carbon films support grid for carrying the sample suspension is moved into glass training
It supports in ware, closes the lid, be then put into togerther in vacuum oven together with culture dish dry.
11. preparation method as claimed in claim 10, which is characterized in that vacuum degree < 10 in the vacuum oven-3Pa,
Heating temperature≤60 DEG C, 30~40min of drying time.
12. preparation method as claimed in claim 11, which is characterized in that the heating temperature is 60 DEG C.
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